Abstract 154: Direct Activation of Type I Protein Kinase A (PKA) by Oxidants Independently of cAMP is Mediated by RI Subunit Interprotein Disulphide Bond Formation
Isolated rat hearts or isolated ventricular myocytes were treated with H2O2 (1μM-10mM). This caused a dose-dependent increase in interprotein disulfide bond formation between PKAs two regulatory RI subunits observed on non-reducing immunoblots. Control myocytes contain 14% disulfide PKA dimer, increasing to 68% (p<0.05) with H2O2 (100μM, 5min). We hypothesised this may have a functional correlate. Fractionation showed the RI disulfide translocated from cytosol to membrane and myofilament/nuclear enriched fractions. This was confirmed by confocal immunofluorescent imaging of RI in myocytes, with enhanced filament and nuclear staining only present in H2O2 cells. H2O2 also increased phosphorylation of multiple proteins, including a 25kDa (4.5 fold, p<0.05) and a 10kDa protein (19 fold, p<0.05), as detected with a pan PKA substrate phosphospecific antibody. These proteins were identified and the increases confirmed using other phospho antibodies specific to PKA sites on phospholamban and troponin I. The functional result of H2O2 (100μM) treatment in cardiac myocytes was increased amplitude of contraction to 0.204μm, compared to 0.111μm in controls (p<0.05). However, H2O2 did not elevate cAMP at any dose (1μM-1mM). H2O2-induced substrate phosphorylation and increased contraction was blocked by the PKA inhibitor H89 (10μM). H2O2 also caused a dose dependent decrease in Type I PKA holoenzyme complex size, as evidenced by immunoblot analysis of the catalytic subunit in fractions eluting from a Superose 12 gel filtration column, further supporting activation without cAMP. PKA R subunits (which have AKAP binding sites) were affinity purified from hearts with cAMP-agarose and analysed by Coomassie stained SDS-PAGE. One protein was present only in H2O2 samples and identified by LC-MS/MS as α myosin heavy chain (αMHC). This interaction was confirmed by immunoprecipitating αMHC, which co-purified with RI PKA in a H2O2 dose dependent manner. In vitro binding assays demonstrated the interaction only occurred when RI was in the disulfide oxidised state. We conclude that Type I PKA is redox sensitive and is activated directly by H2O2 by a mechanism involving interprotein disulfide formation, with αMHC acting as an AKAP to target it to myofilament substrates.